Enzyme sensor, in which the high specificity of an enzyme toward its substrate is used, is found to be useful for measurement of several compounds, and the enzyme sensor has already been practiced in quantitative analyses of glucose, etc. The enzyme sensor now practiced, in which an enzyme possessing high specificity toward a substance to be measured is immobilized, is used for determination of an object substance in a sample, by electrochemically detecting an amount of hydrogen peroxide formed, or of oxygen consumed when an enzyme acts on an object substance in a sample. Accordingly, enzymes to be employed in enzyme sensors of this type are restricted to oxidation enzymes, so-called "oxidases," which form hydrogen peroxide.
Usually, oxidases, which selectively oxidize substances to be measured, are separated from microorganisms selected through screening and are purified for use. However, a microorganism which produces an object enzyme is not always found through the screening process, and even if it is found, there are many cases where the microorganism cannot be employed owing to enzymatic properties, such as substrate specificity, the K.sub.m value, and stability. In addition, because of low productivity by microorganisms or difficult separation and purification, there are many cases where enzyme sensors do not come into practical use, and an enzyme sensor serving for practical use is only a glucose sensor under the present situation.
Concerning oxidase, an enzyme sensor is developed for measurement of ethanol by use of an alcohol oxidase originated in yeast, and its practical use is examined in the system of using a hydrogen peroxide electrode or an oxygen electrode, but actually, it does not bring about a commercial success. This is because substrate specificity of the enzyme is low and a life-time of the enzyme is considerably short.
On the other hand, as enzyme other than the oxidases above described, there are dehydrogenases which donate electrons occurring in the oxidation process to prosthetic groups, such as PQQ, FAD, NAD, and NADP, being not always accompanied by oxygen consumption nor hydrogen peroxide generation. In this type of enzymes, there are greater number of types than are oxidases. Of the dehydrogenases, the presence of enzymes suitable for sensor is also known.
Accordingly, the present inventors developed a new enzyme sensor with a substrate specificity higher than, and with a stability superior to, the sensor by means of enzyme originated in yeast in the preceding publication (Japanese Patent Application No. 253,850/87). The enzyme used in the sensor was an alcohol dehydrogenase having PQQ (pyrroloquinoline quinone) as a prosthetic group originated in acetic acid bacteria. By this invention, a sensor superior to the sensor by means of an enzyme originated in yeast was developed, resulting in one success.
However, dehydrogenases other than alcohol dehydrogenases have hardly been employed yet as a source of enzymes for the sensor. This is because the coenzymes, i.e. PQQ, FAD, NAD, and NADP, are of high prices, which leads to high analysis costs. In order to solve the problem, it was attempted, for example, to immobilize NAD and to regenerate for use, which does not come into practical use yet, though. On the other hand, a method employing an artificial electron acceptor as a mediator (an electron transport intermediate) is developed, thereby preparing an enzyme sensor for a high specificity toward glucose, etc., as well as toward ethanol, and its practical use is examined. However, it is evident that the cost is high even if an artificial mediator is employed, and mediators usually employed are usually coloring substances, so that the waste fluids are colored, to cause a problem of waste water treatments if discarded as it is. In addition, a high-priced mediator is wasted every measurement, thereby resulting in a further higher cost, so that the improvement has been desired.
It is proposed that an artificial mediator in the form of a thin film is applied to the surface of an electrode, which is then coated with an enzyme, followed by being further covered with a semipermeable membrane (EP 78636B1). In other methods, it was proposed that a mediator hardly soluble in water is incorporated into a electrode material (Agric. Biol. Chem., 52, 1557, (1988), and that in the case of a highly water-soluble mediator, first the mediator is added to an electrode, then a thin film is made of a mixture comprising an ionic high molecular compound and an enzyme, so as not to elute the mediator into an electrolyte (Agric, Biol. Chem., 52, 3187 (1988)). However, in both of the cases, it is troublesome to prepare the electrodes, and the enzyme is employed so as to form a thin film, after the incorporation of mediator. In the latter case, there is no examination of employment of dehydrogenase.
As earlier described, conventional enzyme-modified electrodes have several problems; the preparation is troublesome; very difficult operations are required for mass-production of quality-controlled product; in addition, their life-time for repeating use is short; and enzymes that can be utilized are limited. Accordingly, instead of the conventional troublesome process of successive covering of the thin membrane layer of a mediator, the thin membrane layer of enzyme, and the layer of a semipermeable membrane with the surface of an electrode, the present inventors made an electrode material with a homogeneous composition from electron carriers, such as graphite carbon pastes, which are usually employed as an electrode material, an enzyme, and a mediator, by the addition of suitable vehicles like liquid paraffin, by mixing them to a homogeneous composition thereby the surface of an electrode substrate (e.g. carbon electrode) being covered with the electrode material (referred to as an enzyme-modified electrode material), and by this considerably easy method, the present inventors established a process of the preparation of an enzyme-modified electrode for respective enzymes, to complete the present invention after several researches for the purpose of providing the enzyme-modified electrode-incorporated sensor excellent in properties.